The objective of this Phase II project is to fully develop and characterize the performance of nano-thin polyimide membrane sample supports for Transmission Electron Microscopy (TEM) and to design, test, and implement batch- to mass-production techniques in order to bring them to a competitive market. The fundamental aim is to produce a new tool for microscopists. This innovation 's unobstructed TEM sample substrates with millimeter-scale apertures and no intervening grid bars - will be highly useful for TEM scientists and technicians. Significantly, the unobstructed open aperture will provide dramatic scientific advantage for those working in the cellular tomography, cryoelectron microscopy, and structural biology fields that are returning important information and images of cells and subcellular components in their native state. The 2003 NIH Roadmap initiative, New Pathways to Discovery, recognizes the need for better tools for biomedical research and cites structural biology as one field presenting today's major research challenges. The Phase I project demonstrated that nano-thin polyimide membranes are strong and stable over unobstructed millimeter-scale apertures (and long beam exposure) thus demonstrating the feasibility of gridless TEM supports. In addition, the sample supports proved robust to cryogenic processing and typical electron microscopy specimen preparation protocols. The Phase II project will focus on full product and manufacturing process development, guided by four technical objectives: 1) reproduce polyimide TEM supports developed during Phase I as a baseline for the Phase II studies, 2) characterize the supports in terms of physical properties and application performance in cooperation with researchers in the field, 3) continue development of large scale production techniques, and 4) evaluate results from all testing and manufacturing efforts and apply these insights to optimize the product and production methods. A successful Phase II effort will result in two types of specialized sample supports for microscopy: 1) the gridless support with large (2mm) unobstructed apertures and ease of use similar to conventional metal grids, and 2) a composite support blending the strength of nano-thin polyimide membranes and custom-designed small cross-section grid wires specifically for the high-tilt imaging requirements of cryoelectron microscopy of vitrified sections (CEMOVIS). In both cases, the reliability of these sample supports will be a particular benefit where loss of irreplaceable or high-value samples must be avoided. Gridless TEM Sample Supports This project to develop specialized sample holders for transmission electron microscopy is aligned with NIH Roadmap Initiative, New Pathways to Discovery. The sample holders will enhance the scientific return for researchers in structural biology, especially those studying the cell, and imaging cells and cellular structures in their native state. [unreadable] [unreadable] [unreadable]